Induced expression of rolC for study of its effect on the expression of genes associated with nicotine synthesis in tobacco

Background. Agrobacterium rhizogenes rol genes cause not only hairy root syndrome in plants, but also affect their secondary metabolism. There are cases of increasing of nicotine content in transgenic tobacco roots expressing rolC alone or in combination with other rol genes. In this work, we evaluated the change in the expression of nicotine synthesis genes and their regulators in response to the induction of expression of rolC . Materials and methods. Plant material was represented by three Nicotiana tabacum genotypes: cv. Samsun and two transgenic lines, derived from this cultivar and containing rolC under dexamethasone inducible promoter: A. rhizogenes rolC (Pdex-A4 rolC ) and N. tabacum rolC (Pdex-t rolC ) correspondingly. Fluidigm Biomark RT-PCR was used for evaluation of expression of QPT1 , QPT2 , A622 , ODC , ADC , PMT1 , PMT2 , PMT3 , PMT4 , MPO1 , MPO2 , BBL , MATE1 , MATE2 , ARF6 , ERF168 , ERF189 , A4 rolC , Nt rolC , and reference gene gapdh . HPLC-MS / MS analysis was used to determine content of nicotine and its derivatives in plant tissues. Results. Expression of PMT genes for the synthesis of the pyrrolidine ring, as well as the genes, controlling enzyme for final stages of nicotine synthesis, was higher in transgenic lines without induction of rolC expression. Regulatory genes were activated by dexamethasone in both transgenic and control lines, indicating the inapplicability of rolC dexamethasone induction for their study. The level of expression of PMT and MPO genes increased over time in transgenic dexamethasone-induced lines. Nicotine content decreased in transgenic dexamethasone-induced plants. Conclusions. The rolC gene does not play a primary role in the regulation of nicotine synthesis genes. The mechanism of regulation of different nicotine biosynthesis genes and TFs varies.